•Improved protocol for the separation of glycolipids: SQDG, MGDG and DGDG.•Marine glycolipids are successfully separated and quantified by the new protocol.•New approach extends the power of TLC–FID enabling analysis of 18 lipid classes. We demonstrate improved power of Iatroscan thin layer chromatography/flame ionization detection (TLC–FID) technique for analysis of complex marine lipid mixture by developing protocol for the separation and analysis of glycolipids including sulfoquinovosyldiacylglycerols (SQDG), monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG). We have modified the common protocol used so far for the analysis of lipid classes by replacing the elution step which uses pure acetone for the elution of acetone mobile polar lipids, with the elution step containing chloroform–acetone (72:28, v:v) for separation of MGDG and DGDG. To separate SQDG from the complex lipid matrix we introduced solvent mixture acetone–chloroform–methanol–formic acid (33:33:33:0.6, v:v:v:v). Quantification of glycolipid classes was performed after calibration with glycolipid standards for the masses between 0.2 and 2.7–5.0μg. With this new protocol we have successfully separated three glycolipids from the complex particulate lipid mixture of the seawater samples. Such an approach extends the power of existing protocol for the analysis of lipids which altogether ensure detection and quantification of 18 lipid classes what was demonstrated on seawater samples. This enables to gain a very broad system overview of the particularly complex environments as are seas, oceans and freshwaters.